The present invention is concerned with a new group of cyclopentane and cyclopentene compounds and their use as diagnostic agents for detecting influenza A and B. The compounds of the present invention bind to influenza A and B neuraminidase. Moreover, these compounds possess functionality which allows them to be bound to a surface or to a detectable label.
The diagnostic method of the present invention depends upon the ability of the disclosed compounds to bind specifically to the active site of influenza virus neuraminidase, or functionalized derivatives of such compounds, as binding and/or detecting agents to identify influenza virus in clinical specimens. The term xe2x80x9cneuraminidase bindersxe2x80x9d is used hereinafter to refer to these compounds and their functionalized derivatives. The method and compounds of the present invention can function either in the presence or the absence of compounds binding non-specifically to influenza virus neuraminidase.
Influenza A and B viruses are major causes of acute respiratory disease, with an estimated 30-50 million infections annually in the United States alone. Influenza A has been responsible for major epidemics, such as the xe2x80x9cSpanish fluxe2x80x9d of 1919 which killed millions of people. Many viral and bacterial infections may exhibit symptoms similar to those of influenza. The rapid identification of respiratory viruses would enable physicians to use the most appropriate therapy early in the illness. For example, an early and accurate diagnosis would allow decisions regarding the use of antibacterial therapy and hospitalization of children and the elderly.
Laboratory tests for the identification of viruses in clinical material are widely used, and a variety of different detection methodology is available. The textbook, Laboratory Diagnosis of Viral Infections, Marcel Dekker, 1992, Ed. E. H. Lennette, generally discusses methods which are used for a wide range of viruses, including influenza virus.
A number of tests are available for the diagnosis of influenza A and B. The traditional method of identifying influenza viruses has been the use of cell culture, which is highly sensitive and specific. Unfortunately, the time required for culture, isolation and identification of influenza virus can range between 2 and 10 days, thus making it virtually useless in guiding the physician to an appropriate therapy. Since influenza virus infection is normally self-limited, diagnosis must be rapid if therapy is to be effective. In other words, such cell culture methods are normally only of value in providing retrospective epidemiological information.
In addition to the cell culture methods for detecting influenza, there have recently become available a few rapid direct tests, which are specific for influenza A. Thus, a monoclonal immunofluorescence assay (IFA) has been reported (Spada, B. et al., J. Virol. Methods, 1991, 33: 305) and at least one enzyme immunoassay (EIA) is available (Ryan-Poirier, K. A. et al., J. Clin. Microbiol., 1992, 30: 1072). A number of comparisons of these rapid detection methods for influenza A have been reported; see for example Leonardi, G. P. et al., J. Clin. Microbiol., 1994, 32: 70, who recommended that direct specimen testing be used together with culture isolation, so as to permit both identification of the virus in time to institute therapy and infection control measures, and to monitor the antigenic constitution of influenza strains prevalent in the community for epidemiological purposes. The IFA method is reported to be labor-intensive, and requires considerable technical expertise, with the results often being difficult to interpret. On the other hand, the EIA method (Directigen FLU-A; Becton Dickinson Microbiology Systems) give a high level of false-positive results, and it has been recommended that this assay be used in laboratories only in addition to or as a substitute for direct immunofluorescence tests (Waner, J. L. et al., J. Clin. Microbiol., 1991, 29: 479) .
As well as the problems mentioned above with the available rapid assays for influenza, there are other fundamental deficiencies in some of these methods. Firstly, none of the available assays can detect influenza B, which means that even a negative test result would leave the physician uncertain about the type of therapy that should be used. Secondly, if a rapid immunoassay method depends on the use of antibodies to one of the influenza A proteins, there may be a serious problem in detecting new strains of the virus which have undergone a drift or shift in the structure of the antigenic proteins. Influenza A is notorious for its propensity to undergo such changes.
Neuraminidase is one of the key proteins present on the surface of the influenza virus, and it plays an important role in the ability of the virus to infect human cells. It has long been thought that agents which bind to the neuraminidase enzyme might prevent invention by influenza, and much effort has gone into seeking such binders. While many compounds have shown in vitro activity against influenza neuraminidase, only recently has it been established that it is possible to achieve protection from influenza infection in vivo by the use of a powerful neuraminidase binder which binds to the active site of the enzyme (see von Itzstein, M. et al., Nature, 1993, 363: 418 and International Patent Applications No. WO 92/06691 and WO 91/16320). In particular, it has been found that 2,3-didehydro-2,4-dideoxy-4-guanidinyl-N-acetylneuraminic acid (Compound I, designated GG167) is a potent binder of influenza neuraminidase, and also shows potent in vivo antiviral activity in animals (Ryan, D. M. et al., Antimicrobiol Agents and Chemotherapy, 1994, 38: 2270) and in human volunteers (Hayden, F. G. et al., J. American Medical Assoc., 1996, 275: 295). 
More recently, it has been found that certain substituted cyclohexene derivatives of sialic acid are also potent binders of influenza virus neuraminidase (Kim, C. U. et al., J. Amer. Chem. Soc., 1997, 119: 681), and specifically the compound (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (GS 4071).
It is the purpose of the present invention to provide a simple and sensitive means for detecting influenza viruses.
The present invention is concerned with cyclopentane and cyclopentene compounds represented by the following formulae: 
wherein:
U is CH, O or S;
Z is xe2x80x94C(R2)R3), xe2x80x94CHxe2x80x94N(R2)(R3), C(R3)[(CH2)nR2], or CHxe2x80x94C(R3)(CH2)nR2;
R1 is H, (CH2)nOH, (CH2)nNH2, (CH2)nNR10R11, (CH2)nOR11, (CH2)nSR11, or (CH2)n halogen;
R9 is (CH2)nCO2H, (CH2)nSO3H, (CH2)nPO3H2, (CH2)nNO2, esters thereof, or salts thereof;
R2 is H, NHC(O)R5, NHC(S)R5, NHSO2R5, C(O)NHR5, SO2NHR5, CH2S(O)R5, or CH2SO2R5;
R3 is H, (CH2)nCO2R10, (CH2)mOR10, C(O)N(R10)m, (CH2)nN(R10)m, CH(R10)m, (CH2)n(R10)m, CH2CH(OR10)CH2OR10, CH(OR10)CH(OR10)CH2OR10, CH2OR10, CH(OR10)CH2NHR10, CH2CH(OR11)CH2NHR10, CH(OR10)CH(OR10)CH2NHR10, C(xe2x95x90NR10)N(R10)m, NHR10, NHC(xe2x95x90NR10)N(R10)m, (CH2)m-Xxe2x80x94Wxe2x80x94Y, CH2CH(Xxe2x80x94Wxe2x80x94Y)CH2OR10, CH(Xxe2x80x94Wxe2x80x94Y)CH(OR10)CH2OR10, CH(Xxe2x80x94Wxe2x80x94Y)CH2(OR10), CH(OR10)CH(Xxe2x80x94Wxe2x80x94Y)CH2OR11, CH(OR10)CH2(Xxe2x80x94Wxe2x80x94Y), CH2CH(Xxe2x80x94Wxe2x80x94Y)CH2NHR10, CH(Xxe2x80x94Wxe2x80x94Y)CH(OR11)CH2NHR10, CH(Xxe2x80x94Wxe2x80x94Y)CH2(NHR10), CH(OR10)CH(Xxe2x80x94Wxe2x80x94Y)CH2NHR11, or CH(NHR10)CH2(Xxe2x80x94Wxe2x80x94Y);
R4 is H, (CH2)nOH, (CH2)nNR10R11, (CH2)nNH2, (CH2)nC(xe2x95x90NH)(NH2), (CH2)nNHC(xe2x95x90NR11)NH2, (CH2)nNHC(xe2x95x90NR7)NH2, (CH2)nCN, (CH2)nN3, C(xe2x95x90NH)NH2, C(NR7)NH2, or C(NR11)NH2;
R5 is H, lower alkyl, branched chain alkyl, cyclic alkyl, halogen substituted alkyl, aryl, substituted aryl, or CF3;
R7 is H, (CH2)nOH, (CH2)nCN, (CH2)nNH2, or (CH2)nNO2;
R10 is H, lower alkyl, lower alkylene, branched alkyl, cyclic alkyl, (CH2)n aromatic, (CH2)n substituted aromatic, or when m is 2 both R10 groups can also be interconnected to form an N substituted heterocyclic ring, or other 5 or 6 membered heterocyclic ring;
R11 is lower alkyl, branched alkyl, (CH2)m aromatic, or C(O)OR10;
R12 and R13 is H, (CH2)nOH, (CH2)nNH2, (CH2)nNR10R11, (CH2)nOR11, (CH2)nF, (CH2)nOC(O)R11, (CH2)nNHC(O)R11, or Xxe2x80x94Wxe2x80x94Y;
m is 1 or 2;
n is 0-4;
p is 0 or 1;
X is O, S, CH2, or NH;
W is a spacer group made up of a chain of 4 to 100 atoms, and optionally also comprising of substituted carbon and/or nitrogen atoms and optionally including oxygen or sulphur atoms;
Y is OH, SH, NH2, CHxe2x95x90O, CHxe2x95x90CH2, CO2H, CONHNH2, or NH-biotinyl, or a protected form of one of these end functionalities.
It has been found according to the present invention that the compounds of the present invention can be used to detect influenza virus by selectively binding the influenza virus and by being able to attach to a surface or to a detectable linking group. Therefore, another aspect of the present invention relates to a method for detecting influenza virus. The method comprises the step of exposing a sample suspected to comprise the influenza virus to at least one of the above-disclosed compounds.
Still other objects and advantages of the present invention will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described only the preferred embodiments of the invention, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.
The cyclopentane and cyclopentene compounds of the present invention are represented by the following formulae: 
wherein:
U is CH, O or S;
Z is xe2x80x94C(R2)R3), xe2x80x94CHxe2x80x94N(R2)(R3), C(R3)[(CH2)nR2], or CHxe2x80x94C(R3)(CH2)nR2;
R1 is H, (CH2)nOH, (CH2)nNH2, (CH2)nNR10R11, (CH2)nOR11, (CH2)nSR11, or (CH2)n halogen;
R9 is (CH2)nCO2H, (CH2)nSO3H, (CH2)nPO3H2, (CH2)nNO2, esters thereof, or salts thereof;
R2 is H, NHC(O)R5, NHC(S)R5, NHSO2R5, C(O)NHR5, SO2NHR5, CH2S(O)R5, or CH2SO2R5;
R3 is H, (CH2)nCO2R10, (CH2)mOR10, C(O)N(R10)m, (CH2)nN(R10)m, CH(R10)m, (CH2)n(R10)m, CH2CH(OR10)CH2OR10, CH(OR10)CH(OR10)CH2OR10, CH2OR10, CH(OR10)CH2NHR10, CH2CH(OR11)CH2NHR10, CH(OR10)CH(OR10)CH2NHR10, C(xe2x95x90NR10)N(R10)m, NHR10, NHC(xe2x95x90NR10)N(R10)m, (CH2)m-Xxe2x80x94Wxe2x80x94Y, CH2CH(Xxe2x80x94Wxe2x80x94Y)CH2OR10, CH(Xxe2x80x94Wxe2x80x94Y)CH(OR10)CH2OR10, CH(Xxe2x80x94Wxe2x80x94Y)CH2(OR10), CH(OR10)CH(Xxe2x80x94Wxe2x80x94Y)OH2CR10, CH(OR10)CH2(Xxe2x80x94Wxe2x80x94Y), CH2CH(Xxe2x80x94Wxe2x80x94Y)CH2NHR10, CH(Xxe2x80x94Wxe2x80x94Y)CH(OR10)CH2NHR10, CH(Xxe2x80x94Wxe2x80x94Y)CH2(NHR10), CH(OR10)CH(Xxe2x80x94Wxe2x80x94Y)CH2NHR10, or CH(NHR10)CH2(Xxe2x80x94Wxe2x80x94Y);
R4 is H, (CH2)nOH, (CH2)nNR10R11, (CH2)nNH2, (CH2)nC(xe2x95x90NH)(NH2), (CH2)nNHC(xe2x95x90NR11)NH2, (CH2)nNHC(xe2x95x90NR7)NH2, (CH2)nCN, (CH2)nN3, C(xe2x95x90NH)NH2, C(NR7)NH2, or C(NR11)NH2;
R5 is H, lower alkyl, branched chain alkyl, cyclic alkyl, halogen substituted alkyl, aryl, substituted aryl, or CF3;
R7 is H, (CH2)nOH, (CH2)nCN, (CH2)nNH2, or (CH2)nNO2;
R10 is H, lower alkyl, lower alkylene, branched alkyl, cyclic alkyl, (CH2)n aromatic, (CH2)n substituted aromatic, or when m is 2 both R10 groups can also be interconnected to form an N substituted heterocyclic ring, or other 5 or 6 membered heterocyclic ring;
R11 is lower alkyl, branched alkyl, (CH2)m aromatic, or C(O)OR10;
R12 and R13 is H, (CH2)nOH, (CH2)nNH2, (CH2)nNR10R11, (CH2)nOR11, (CH2)nF, (CH2)nOC(O)R11, (CH2)nNHC(O)R11, or Xxe2x80x94Wxe2x80x94Y;
m is 1 or 2;
n is 0-4;
p is 0 or 1;
X is O, S, CH2, or NH;
W is a spacer group made up of a chain of 4 to 100 atoms, and optionally also comprising of substituted carbon and/or ntrogen atoms and optionally including oxygen or sulphur atoms;
Y is OH, SH, NH2, CHxe2x95x90O, CHxe2x95x90CH2, CO2H, CONHNH2, or NH-biotinyl, or a protected form of one of these end functionalities.
The lower alkyl groups contain 1 to about 8 carbon, and preferably 1 to about 3 carbon atoms, and can be straight, branched-chain or cyclic saturated aliphatic hydrocarbon groups.
Examples of suitable alkyl grops include methyl, ethyl and propyl. Examples of branched alkyl groups include isopropyl and t-butyl. Examples of suitable cyclic aliphatic groups typically contain 3-8 carbon atoms and include cyclopentyl and cyclohexyl. The aromatic or aryl groups are preferably pehnyl or alkyl substituted aromatic groups (aralkyl) such as phenyl C1-3 alkyl such as benzyl.
Examples of substituted cycloalkyl groups include cyclic aliphatic groups typically containing 3-8 carbon atoms in the ring substituted with alkyl grops typically having 1-6 carbon atoms and/or hydroxy group. Usually 1 or 2 substituted groups are presented.
The esters are typically lower alkyl esters having 1 to about 12 carbon atoms and preferably 1 to about 3 carbon atoms and aryl esters containing 6 to 14 carbon atoms. The alkyl esters can be straight-chain, branched-chain or cyclic saturated aliphatic hydrocarbons.
Examples of some alkyl esters are methyl, ethyl, propyl, isopropyl, t-butyl, cyclopentyl and cyclohexyl esters. The aryl esters are preferably phenyl or alkyl substituted aromatic esters (alkaryl) including C1-3 alkyl substituted phenyl such as benzyl.
The lower alkylene group can be straight, branched chain or cyclic unsaturated hydrocarbon grop and contains 2-8 carbon atoms and preferably 2-3 carbon atoms. Examples of alkylene grops are vinyl, 1-propenyl, allyl, isopropenyl, 2-methyl-2-propenyl and cyclopentenyl.
The N-heterocyclic rings contain 3-7 atoms in the ring. The heterocyclic rings can be substituted such as with a lower alkyl grop. Examples of suitable heterocyclic groups are pyrrolidino, methylpiperidino and 2-ethylpiperidino.
Suitable spacer groups W include, but are not limited to, linear peptides, oligosaccharides, polyols, polyethylene glycol groups, hydrocarbon groups and hydrocarbon groups linked together with oxygen or sulphur atoms or with carbonyl, amido, urea or hydrazide functionalities. Spacer groups W may also comprise combinations of these various groups.
Suitable protecting groups for the end functionality Y include, but are not limited to, esters of the OH, SH and CO2H groups, carbamates of the NH2 and CONHNH2 groups, and acetals of the CHxe2x95x90O group.
As used herein, the term xe2x80x9chydrocarbon groupxe2x80x9d includes saturated and unsaturated straight or branched hydrocarbon groups, including aryl groups, and combinations of such groups.
Pharmaceutically acceptable salts of the compounds of formula (I) include those derived from pharmaceutically acceptable, inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic, trifluoroacetic and benzenesulphonic acids.
Salts derived from appropriate bases include alkali such as sodium and ammonia.
The compounds of the present invention bind relatively strongly to influenza virus neuraminidase, with IC50 of 10 xcexcM or better.
Compounds of the present invention can be prepared by a variety of methods. By way of illustration purposes, synthesis of some of the preferred compounds is given below: 
In the formula (I)
R2=H;
R9=CO2H;
R4=NH2, or NHC(xe2x95x90NH)NH2;
Z=CHxe2x80x94CH(NHCOCH3)-1-ethyl-propyl;
U=CH;
P=0;
R12=Xxe2x80x94Wxe2x80x94Y;
R13=H;
X is OC(O)NH;
W is (CH2)6, (CH2)6NHCONH(CH2)6, (CH2CH2O)2CH2CH2, (CH2)6(NHCOCH2)3, (CH2)6NHCO(CH2)11, (CH2)6NHCO(CH2)5, (CH2)6[NHCO(CH2)5]4, (CH2)6[NHCO(CH2)5]NHCOCH2(OCH2CH2)16, (CH2)6[NHCO(CH2)5]2, (CH2)6[NHCO(CH2)5]4NHxe2x80x94COCH2CH2, or (CH2)6NHCOCH2CH2;
Y is NH2, NH-Biotin, CONHNHBoc, NHBoc, CONHNH2, or CO2H. 